Because the Y chromosome is present in just one copy and only in males, it faces a diminished capacity to purge deleterious mutations. Our understanding of the complete set of functional elements on the Y chromosome lags far behind the rest of the genome, and this is especially true for the Drosophila model system. There are now 12 complete genome sequences for species of Drosophila whose well-known phylogeny provides a rich resource for analysis of Y chromosome dynamics. A major recent finding is that among these 12 species the gene gains largely outnumber gene losses, which is at odds with the view that the Y is simply degenerating. In this proposal, we aim first, to complete the annotation of Y-linked genes across the Drosophila species whose genomes have been sequenced. We have devised and perfected a highly efficient Illumina/Solexa-sequence based method for identifying Y-specific contigs, and plan to use it to identify, assemble, and annotate the Y-linked contigs and their embedded genes. RNAi knock-down will be used to identify the genes on the D. melanogaster and D. pseudoobscura Y that are essential for male fertility. Second, we will quantify and model intraspecific polymorphism in protein- coding genes on the Y chromosome of D. melanogaster, D. simulans, and D. pseudoobscura, as well as interspecific divergence among the 12 species. Analysis of polymorphism and divergence has started to place bounds on the levels of background selection acting on Drosophila Y chromosomes. The roles of Muller's ratchet, hitchhiking by selective sweeps, and the Hill-Robertson effect will also be assessed by fitting models of sequence evolution to site frequency data using approximate Bayesian computation. D. simulans is included as a second and more polymorphic cosmopolitan species to compare to melanogaster, and D. pseudoobscura's Y chromosome is derived within the past 18 Myr from formerly autosomal sequences. Third, we will assemble and finish the Y-to-autosome translocated region of D. pseudoobscura genome. This is motivated by the translocation of heterochromatic, Y-linked genes onto the dot chromosome in this species, presenting a fortuitous opportunity to study the effect of the loss of male-restricted transmission and the loss of heterochromatin in this region. This project will entail BAC sequencing, RT-PCR tests of expression, and analysis of polymorphism in this region. Finally, the fourth aim is to characterize and quantify functional variation associated with the Y chromosome. Simple population genetic models predict rapid fixation of favored Y chromosomal alleles, and yet there is compelling evidence that functional polymorphism is maintained. Theory further shows that interactions between Y and other chromosomes are more likely to be able to retain polymorphism, and recent work has demonstrated large effects of Y-linked variation on gene expression throughout the genome. Experiments designed specifically to quantify the role of epistasis in Y chromosomal evolution are well motivated. PUBLIC HEALTH RELEVANCE: The Y chromosome of Drosophila provides an ideal system for testing many concepts related to the evolution of sex chromosomes. The proposed study entails primary discovery of most of the Y- linked genes across a group of 12 species and a detailed study of their evolutionary divergence.